Starting with established lines of mouse and Chinese hamster cells, we have isolated mutants of the following types: (1) defective in the A system of amino acid transport; (2) loss of affinity for alpha-aminoisobutyric acid (AIB) by the A system; (3) defective in leucine uptake; (4) increased net uptake of leucine. We have also developed an improved procedure for the isolation of membrane vesicles that actively transport AIB. During the second year of the grant, we will make vesicles from the normal and mutant cell lines, and analyze their transport activities. Differences between the mutant and normal vesicles will then be used as the basis for reconstitution experiments, in which membrane proteins from active cells will be used to restore activity to vesicles from inactive cells.